T lymphocytes, i.e., T cells, are central players in the immune response by virtue of their ability to recognize antigens with a high degree of specificity, to act as effector cells in the lysis of specific target cells, and to regulate the nature and intensity of the immune response. Once research clarified the role of T lymphocytes in the immune system, it became useful to stimulate selected lymphocyte populations and subpopulations that could play a role in immune responses, especially in response to tumors.
Interleukin-2 (IL-2), a lymphokine produced by helper T cells, stimulates the growth of T cells and NK cells that bear IL-2 receptors, either in vivo or in vitro. The in vitro incubation of resting lymphocytes in media containing IL-2 for three to four days induces the generation of lymphocytes capable of lysing fresh tumor cells, but not normal cells. These lymphocytes are referred to as lymphokine activated killer (LAK) cells. See, for example, I. Yron et al., J. Immunol., 238 (1980); M. T. Lotze et al., Cancer Res., 41, 4420 (1981); and S. A. Rosenberg et al., Natl. Cancer Inst., 75, 595 (1985). This ability of IL-2 to induce the proliferation of lymphocytes with immune reactivity and with the ability to lyse fresh autologous, syngeneic, or allogeneic natural killer (NK) cell resistant tumor cells, but not normal cells, has resulted in further developments in the area of adoptive immunotherapy, i.e., cell transfer therapies.
Typical adoptive immunotherapy involves the administration of immunologically active cells to an individual for the purpose of providing a beneficial immunological effect to the individual, e.g., reduction or control of cancerous or diseased tissue. These immunologically active cells, e.g., lymphokine-activated killer cells and tumor infiltrating cells, are typically taken either from the individual to be treated, known as an autologous treatment, or from another individual, known as an allogenic treatment. Lymphokine-activated killer cells are typically taken by venipuncture or leukophereses. Tumor infiltrating cells are taken from tumors removed during surgery. The lymphocytes are cultured to increase their number and activate their antitumor activity, and infused back into the patient. Thus, the majority of conventional efforts in adoptive immunotherapy are directed at increasing the number of activated cells in vitro followed by infusion back into the patient.
Animal experiments transferring immunologically active cells, e.g., LAK cells, from healthy animals to animals with cancerous tumors have suggested that adoptive immunotherapy can elicit an antitumor effect in certain tumor models with a high degree of effectiveness. Furthermore, the administration of IL-2 in addition to LAK cells has proven effective in the treatment of a variety of murine malignancies. IL-2 also leads to the in vivo proliferation of transferred LAK cells. These initial animal studies were repeated with humans in clinical trials.
The human studies demonstrated that LAK cells plus IL-2, or IL-2 alone, can be effective in mediating the regression of established metastatic cancer in selected patients. See, for example, S. A. Rosenberg, "Immunotherapy of Patients with Advanced Cancer Using Interleukin-2 Alone or in Combination With Lymphokine Activated Killer Cells" in Important Advances in Oncology 1988, J. B. Lippincott Co., 217, (1988).
Although adoptive immunotherapy has met with certain success, a difficulty with many of the experimental protocols is that a large number of cells is required in the therapy. Furthermore, conventional protocols have proven less than desirable because of the large amount of culture medium, the large amount of IL-2, the large number of hours involved in culturing cells to develop LAK activity, the time involved in clinical treatment, and the side effects of treatment. The infusion of LAK cells also frequently results in the development of respiratory difficulty. These cells are large and sticky and bind to the vascular system in the lungs causing severe damage that can result in the patient requiring intensive care management.
To overcome some of these difficulties, advances have been made to improve the in vitro culturing process. For example, T cells cultured in the presence of IL-2 and monoclonal antibodies (MoAb) against the antigen receptor complex CD3, i.e., anti-CD3 MoAb, have been found to proliferate and demonstrate LAK activity on a per cell basis. See, for example, P. M. Anderson et al., Cancer Immunol. Immunother., 27, 82 (1988); P. M. Anderson et al., J. Immunol., 142, 1383 (1989); and A. C. Ochoa et al., Cancer Res., 49, 963 (1989).
Some efforts have been aimed at activating in vivo antitumor mechanisms; however, there has been limited success in this area. For example, patients have received high doses of IL-2 with significant toxicity. The direct infusion of anti-CD3 monoclonal antibody alone induces nonspecific antitumor function in mice. See, for example, D. W. Hoskin et al., Cancer Immunol. Immunother., 29, 226 (1989). Based on the positive results in murine models, direct infusion of anti-CD3 MoAb has been attempted in humans. Although patients who have directly received the anti-CD3 MoAb OKT3 have experienced the activation of some T cells in vivo. the toxicity of intravenous OKT3 reaches the maximum tolerated dose (MTD) before immune efficacy develops. It is believed that the free OKT3 is responsible for the majority of these toxic effects. In addition, the infusion of anti-CD3 induces the production of endogenous antibodies which neutralize the effect of the anti-CD3 thus complicating the possibility for repeated therapy.
Although direct infusion of anti-CD3 results in significant levels of toxicity, cells activated by anti-CD3 outside of the body are useful in adoptive immunotherapeutic techniques. Anti-CD3 induces, at least in part, activation of tumoricidal T cells as well as activated NK (LAK cells). Conventional IL-2 therapy, however, only activates LAK cells. Furthermore, some studies have shown that certain tumor target cells are more susceptible to anti-CD3 activated T cells than LAK cells. See, for example, J. Stankova et al., Cell. Immunol., 121, 13 (1989).
A comparison of the efficacy of cells stimulated with IL-2 for four days, with anti-CD3 for 72 hours, as well as the antitumor activity induced by the direct infusion of anti-CD3 MoAb has been undertaken. See, for example, S. Gallinger et al., Cancer Res., 50, 2476 (1990). It has been determined that in this murine model the most effective way of reducing tumor is direct infusion of anti-CD3. Infusion of cells stimulated with anti-CD3 was less effective while infusion of cells cultured in IL-2 was least effective.
It has been shown that anti-CD3 induced effector cells acquire cytolytic activity in vitro within 24 hours of incubation of murine lymphocytes with anti-CD3. It has also been shown that in vitro incubation of peripheral blood lymphocytes of normal donors with the anti-CD3 monoclonal antibody OKT3 for 45 minutes resulted in the acquisition of lytic activity against fresh leukemic cells. See, E. Lotzova et al., Nat. Immun. Cell Growth Regul., 6, 219 (1987). It is generally believed, however, that for effective in vivo activity, cells must be incubated with anti-CD3 for at least about 72 hours.
Thus, conventional methods of activating cells in vitro or in vivo have not to date successfully eliminated the need for convenient, effective, and safe therapeutic methods. In general, conventional methods require the development of immunotherapeutic function, e.g., lytic activity, in cells before they are used in adoptive immunotherapy. Because of this limitation, culturing of cells can last for several days before they can be infused back into a patient. A need therefore exists for a more convenient, less time consuming, and more efficient method of culturing cells. That is, a need exists for a method of producing lymphocytes capable of proliferation and enhanced immunotherapeutic efficacy that: (1) is not time consuming; (2) is less expensive; (3) has fewer, if any, side effects; (4) is simple and convenient; and (5) does not require prolonged in vitro culture.